EP1094833B1 - Oxydiertes thymosin beta 4 - Google Patents

Oxydiertes thymosin beta 4 Download PDF

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EP1094833B1
EP1094833B1 EP99913428A EP99913428A EP1094833B1 EP 1094833 B1 EP1094833 B1 EP 1094833B1 EP 99913428 A EP99913428 A EP 99913428A EP 99913428 A EP99913428 A EP 99913428A EP 1094833 B1 EP1094833 B1 EP 1094833B1
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thymosin
oxidised
oxidised thymosin
pharmaceutical formulation
stms
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EP1094833A2 (de
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Robert Duncan Stevenson
Anthony John Lawrence
John Young
Darryl John Cecil Pappin
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University of Glasgow
Cancer Research Horizons Ltd
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Imperial Cancer Research Technology Ltd
University of Glasgow
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57581—Thymosin; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to oxidised thymosin ⁇ 4 isolated from steroid-treated monocytes. More particularly the invention relates to oxidised thymosin ⁇ 4 which can be used to replace steroid therapy.
  • Steroids are effectively used for anti-inflammatory diseases, such as asthma, eczema, allergic reactions, and rheumatic diseases such as rheumatoid arthritis.
  • steroids have serious side effects and are therefore only used in cases where non-steroidal anti-inflammatory drugs are not effective.
  • Monocytes are important immune effector cells that play a fundamental role in cellular immunity.
  • peripheral blood mononuclear cells are also involved in the synthesis and release of a variety of pro-inflammatory enzymes and polypeptide cytokines which modulate neutrophil responses. The production of these components can be suppressed by glucocorticoids and this has been suggested as the basis for their anti-inflammatory action.
  • the neutrophil migration stimulating activity of steroid induced factors suggests that dispersive locomotion tends to prevent cells collecting at a focus and this may be important in terminating inflammatory responses.
  • Chettibi et al (1993, 1994) have investigated the steroid induced stimulatory effect on neutrophil migration using an automated cell tracking assay enabling study of the behaviour of cells migrating on protein-coated glass coverslip. These studies determined:
  • Huff T. et al. (1995) and Heintz D. et al. (1994) describe studies involving beta-thymosins and how they interact with G-actin in a biomolecular complex and inhibit the polymerisation to F-actin under high salt conditions.
  • the oxidised form of thymosin ⁇ 4 is disclosed as inhibiting actin polymerisation, however, only at a 20-fold higher concentration than thymosin ⁇ 4. Neither document however implicates any medical role for oxidised thymosin ⁇ 4. In fact the papers appear to teach away from a positive role for oxidised thymosin ⁇ 4.
  • US5,578,570 discloses a method of treating septic shock by administering thymosin ⁇ 4. There is no disclosure however of oxidised thymosin ⁇ 4 or suggestion that this may have a role in treating septic shock.
  • the present invention is based in part on the observations by the present inventors that the factor associated with neutrophil locomotion is an oxidised form of thymosin ⁇ 4.
  • the present invention provides use of oxidised thymosin ⁇ 4 or physiologically active variant thereof in therapy.
  • oxidised thymosin ⁇ 4 is a form of thymosin ⁇ 4 in which a methionine residue, 6 amino acids from the N-terminus, (Met6), is oxidised such that the residue is converted to methionine sulphoxide.
  • the methionine residue (Met6) may be further oxidised to the methionine sulphone and this as such is also encompassed by the present invention.
  • Other modifications of the methionine residue may also be evisaged, such as complexing the sulphur with metals, which may result in an active form of thymosin ⁇ 4 similar to the oxidised form described herein.
  • oxidised thymosin ⁇ 4 may be obtained for example by reacting native thymosin ⁇ 4 under oxidising conditions, for example by treating with hydrogen peroxide, to form oxidised thymosin ⁇ 4.
  • native thymosin ⁇ 4 may first be obtained and thereafter oxidised to the oxidised form.
  • samples of native thymosin ⁇ 4 may contain low levels, such as 10%, of oxidised thymosin ⁇ 4 thought to be as a result of auto-oxidation.
  • the present inventors are the first to associate the oxidised form of thymosin ⁇ 4 with a physiological activity.
  • the present invention provides the use of purified oxidised thymosin ⁇ 4.
  • preparations of purified oxidised thymosin ⁇ 4 which comprise at least 30%, preferably 60%, more preferably 80%, most preferably 90%, oxidised thymosin ⁇ 4 with the residual portion accounting for non-oxidised thymosin ⁇ 4.
  • the preparations of oxidised thymosin ⁇ 4 comprise substantially all oxidised thymosin ⁇ 4 (ie. substantially no non-oxidised thymosin ⁇ 4).
  • Thymosin ⁇ 4 in an oxidised or non-oxidised form may be obtained from any suitable source, for example from steroid treated monocytes.
  • the thymosin ⁇ 4 may be derived from any suitable species, but is typically of mammalian origin, such as bovine, equine, murine or human origin. It is to be noted that bovine, equine, murine, rat and human thymosin ⁇ 4 are all identical in sequence.
  • bovine thymosin ⁇ 4 may provide a suitable source of thymosin ⁇ 4 for subsequent oxidation and administration to other species, such as humans.
  • physiologically active variants of the oxidised thymosin ⁇ 4 are variants which display the same or similar physiological properties as the oxidised thymosin ⁇ 4. It is to be preferred that such variants would include the oxidised methionine, but may be truncated, deleted or mutated forms thereof.
  • oxidised thymosin ⁇ 4 or physiologically active variants thereof may be used by use of appropriate chemical modification (eg. acetylation) or use of D amino acids.
  • the isolated oxidised thymosin ⁇ 4 may have a blocked N-terminal.
  • a synthetic oxidised thymosin ⁇ 4 comprising the peptide sequence of thymosin ⁇ 4 in oxidised form or physiologically active variant thereof.
  • the synthetic oxidised thymosin ⁇ 4 may be modified and/or amino acid substituted as described above, as long as the physiological activity remains.
  • selenomethionine could be introduced in place of methionine and oxidised in the same manner.
  • the invention further provides the use of an oxidised peptide as described herein in the preparation of a medicament for the treatment of a chronic or acute inflammatory condition.
  • inflammatory conditions include Inflammatory Arthropathies such as Rheumatoid arthritis, Psoriatic arthritis, Crystal arthritis, Reactive arthritis, Ankylosing spondylitis, Infectious arthritis, Juvenile chronic arthritis; Connective Tissue Diseases, such as Systemic Lupus Erythematosis, Sjogren's Syndrome, Polymyalgia Rheumatica, Cranial arteritis; Vasculitic Syndromes, such as Wegener's Granulomatosis, Polyarteritis Nodosa, Churg Strauss Syndrome; Respiratory Diseases, such as Asthma, Chronic Obstructive Pulmonary Disease, Fibrosing Alveolitis, Hypersensitivity Pneumonitis, Sarcoidosis, Allergic aspergillosis, Cryptogenic pulmonary eosinophilia, Bronchiolitis
  • oxidised thymosin ⁇ 4 may be administered in conjunction with other drugs, eg. cytokines such as interferon which may induce an inflammatory response as a side effect.
  • cytokines such as interferon which may induce an inflammatory response as a side effect.
  • oxidised thymosin ⁇ 4 may serve to minimise or reduce physiological or disease states which are characterised in part by inappropriate inflammation.
  • oxidised thymosin ⁇ 4 may be used in the treatment of animals such as cats, dogs, horses, cows, sheep, pigs and goats with similar conditions to those mentioned above.
  • the present invention further provides the use of oxidised thymosin ⁇ 4 in the preparation of a medicament for the treatment of septic shock.
  • oxidised thymosin ⁇ 4 is in a purified form as described above.
  • the invention further provides a pharmaceutical composition comprising oxidised thymosin ⁇ 4 as described herein.
  • the invention further provides use of a nucleotide molecule having a sequence capable of encoding thymosin ⁇ 4 as described herein for subsequently preparing oxidised thymosin ⁇ 4.
  • the invention provides the use of a vector or vectors comprising the nucleotide molecule in the preparation of oxidised thymosin ⁇ 4 and truncated, deleted and mutated forms thereof as described herein.
  • the present invention provides the use of a vector or vectors comprising the nucleotide molecule in the preparation of a medicament comprising oxidised thymosin ⁇ 4 and truncated, deleted and mutated forms thereof for the treatment of a inflammatory condition.
  • oxidised thymosin ⁇ 4 as described herein in place of steroid treatment will alleviate the side effects which are normally associated with the use of steroids.
  • the oxidised thymosin ⁇ 4 can be used for treatment of patients where non steroidal anti inflammatory drugs are currently used as an alternative to steroids because of the risks of side-effects.
  • the amount of oxidised thymosin ⁇ 4 required to be effective in a treatment will, of course, vary and is ultimately at the discretion of the medical or veterinary practitioner.
  • the factors to be considered include the condition being treated, the route of administration, and nature of the formulation, the recipients body weight, surface area, age and general condition, and the particular compound to be administered.
  • a suitable effective dose may lie in the range of about 0.001 to about 120mg/kg bodyweight, e.g. 0.01 to about 120mg/kg body weight, preferably in the range of about 0.01 to 50 mg/kg, for example 0.05 to 20 mg/kg.
  • the total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day or by intravenous infusion for selected duration.
  • the dose range may be about 8 to 9000 mg per day, and a typical dose could be about 50 mg per day. If discrete multiple doses are indicated treatment might typically be 15 mg of oxidised thymosin ⁇ 4 given up to 4 times per day.
  • Formulations of the present invention for medical use, comprise oxidised thymosin ⁇ 4, or a salt thereof together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients.
  • the carrier(s) should be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
  • the present invention therefore, further provides a pharmaceutical formulation comprising oxidised thymosin ⁇ 4 or a pharmaceutically acceptable salt or physiologically functional derivative thereof together with a pharmaceutically acceptable carrier therefor.
  • a method for the preparation of a pharmaceutical formulation comprising bringing into association oxidised thymosin ⁇ 4 or a pharmaceutically acceptable salt or physiologically functional derivative thereof, and a pharmaceutically acceptable carrier therefor.
  • Formulations according to the present invention include those suitable for oral, nasal, topical, vaginal, rectal or parenteral (including subcutaneous, intraarthrodial (ie. within joints) intramuscular and intravenous) administration including biolistic eg. Powderject® administration.
  • Preferred formulations are those suitable for oral, topical or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product into desired formulations.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units as capsules, cachets, tablets, lozenges, comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • Each formulation generally contains a predetermined amount of the active compound; as a powder or granules; or a solution or suspension in an aqueous or non-aqueous liquid such as a syrup, an elixir, an emulsion or draught and the like.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with a binder, (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.
  • a syrup may be made adding the active compound to a concentrated, aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredients.
  • a sugar for example sucrose
  • Such accessory ingredient(s) may include flavourings, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol for example glycerol or sorbitol.
  • Formulations for rectal administration may be presented as a suppository with a conventional carrier such as cocoa butter.
  • Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient.
  • Such formulations suitably comprise a solution of a pharmaceutically and pharmacologically acceptable salt of oxidised thymosin ⁇ 4, that is isotonic with the blood of the recipient.
  • Useful formulations also comprise concentrated solutions or solids containing oxidised thymosin ⁇ 4, which upon dilution with an appropriate solvent give a solution for parental administration as above.
  • the oxidised thymosin ⁇ 4 or physiologically active variant thereof disclosed herein may be administered to the lungs of a subject by any suitable means, but are preferably administered by generating an aerosol comprised of respirable particles, the respirable particles comprised of the active compound, which particles the subject inhales (i.e., by inhalation administration).
  • the respirable particles may be liquid or solid.
  • Particles comprised of oxidised thymosin ⁇ 4 for practising the present invention should include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about .5 to 10 microns in size (more particularly, less than about 5 microns in size) are respirable. Particles of non-respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is preferably minimized. For nasal administration, a particle size in the range of 10-500 ⁇ m is preferred to ensure retention in the nasal cavity.
  • Liquid pharmaceutical compositions or oxidised thymosin ⁇ 4 for producing an aerosol can be prepared by combining the oxidised thymosin ⁇ 4 with a suitable vehicle, such as sterile pyrogen free water.
  • Solid particulate compositions containing respirable dry particles of micronized oxidised thymosin ⁇ 4 may be prepared by grinding dry oxidised thymosin ⁇ 4 with a mortar and pestle, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates.
  • a solid particulate composition comprised of the oxidised thymosin ⁇ 4 may optionally contain a dispersant which serves to facilitate the formation of an aerosol.
  • a suitable dispersant is lactose, which may be blended with the oxidised thymosin ⁇ 4 in any suitable ratio (e.g., a 1 to 1 ratio by weight).
  • Aerosols of liquid particles comprising the oxidised thymosin ⁇ 4 may be produced by any suitable means, such as with a nebulizer. See, e.g., U.S. Patent No. 4,501,729.
  • Nebulizers are commercially available devices which transform solutions or suspensions of the oxidised thymosin ⁇ 4 into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation.
  • compositions for use in nebulizers consist of the oxidised thymosin ⁇ 4 in a liquid carrier, the oxidised thymosin ⁇ 4 comprising up to 40% w/w of the compositions, but preferably less than 20% w/w.
  • the carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride.
  • Optional additives include preservatives if the composition is not prepared sterile, for example, methyl hydroxybenzoate, antioxidants, flavouring agents, volatile oils, buffering agents and surfactants.
  • Aerosols of solid particles comprising the oxidised thymosin ⁇ 4 may likewise be produced with an solid particulate medicament aerosol generator.
  • Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration. Examples of such aerosol generators include metered dose inhalers and insufflators.
  • the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w.
  • the active ingredients may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredients may be formulated in a cream with an oil-in-water cream base.
  • the aqueous phase of the cream may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glyercol and polyethylene glycol and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulphoxide and related analogues.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat.
  • the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax
  • the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyle alcohol, glycerol mono-stearate and sodium lauryl sulphate.
  • the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono-or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like.
  • IL-8 and TNF were purchased from Genzyme, dissolved in phosphate buffered saline (PBS) and stored at 1 ⁇ g per ml at -70°C.
  • Dexamethasone and fMLP were purchased from Sigma Chemical Co. Dexamethasone was prepared as a 10 -2 M stock solution in ethanol and fMLP as a 10 -2 M stock solution in dimethyl sulphoxide (DMSO).
  • Neutrophils were obtained as described by Chettibi et al. [1993]. Briefly, whole blood was mixed with 1:10 v/v of 5% dextran and allowed to sediment at 37°C for 1 hour. The leucocyte rich plasma was layered over Nycoprep 1.077 and centrifuged at 750g for 15 minutes. Erythrocytes in the resulting pellet were removed by hypotonic lysis and neutrophils were washed twice with balanced salt solution (BSS). The cells were checked for viability by trypan blue exclusion (generally greater than 96% viable).
  • Automated cell-tracking migration chambers were made as previously described [Chettibi et al., 1993] and placed on the stage of an inverted phase-contrast microscope within a temperature controlled (37°C) transparent box and locomotion observed by means of a video camera connected to a monochrome monitor and also to an Acorn A5000 computer with a Watford video digitiser programmed to capture and analyze one frame every 5 seconds. Data was obtained from a maximum of 80 selected cells and used to calculate instantaneous speed, the 2-dimensional diffusion coefficient and the locomotion persistence time, using the described procedure to eliminate the contribution of systematic drift [Chettibi et al., 1994].
  • Bovine aorta endothelial cells were cultured on 13mm diameter glass coverslips in a multi-well dish in Dulbecco's modified Eagles medium with 10% foetal calf and 10% horse serum and grown to confluence.
  • Human neutrophils suspended in BSS 0.1% bovine serum albumin (BSA) were labelled with [ 51 Cr]sodium chromate by incubating them at 1 x 10 6 cells/ml for 1 hour, 20 ⁇ Ci/ml with periodic agitation. Free 51 Cr was removed by three washes with BSS 0.1% BSA. 200 ⁇ l of neutrophils were mixed with 800 ⁇ l of STMS peptide factor or other test substances, added to the wells and incubated for 30 minutes at 37°C. Non-adherent cells were washed gently three times with BSS 0.1% BSA and coverslips were placed in a Wilj gamma counter.
  • Neutrophils were stimulated with various agonists for 20 minutes before fixing in 2% buffered gluteraldehyde for one hour and washed twice in PBS. Post-fixation in osmium tetroxide was followed by washing in distilled water. Uranyl acetate was then added to the samples and left in the dark for at least one hour before washing. The cells were passed through a graded series of acetone (or alcohols) ranging from 30% to dried absolute, before critical point drying and mounting.
  • Purified neutrophils were placed on albumin-coated glass coverslips before treatment with various stimuli and incubated at 37°C for 30 minutes.
  • the cells were fixed in 1% paraformaldehyde solution for one hour, washed with BSS and permeabilised with 1% Triton x-100 for 15 minutes at room temperature.
  • Cells were washed three times with BSS and treated with 0.1 mg/ml TRITC labelled phalloidin for 20 minutes at room temperature. Cells were washed three times in BSS at 5 minutes intervals and mounted on glass slides with 50% glycerol. Results were analyzed using confocal microscopy.
  • Filters (Sartorius membrane filter 3 ⁇ m pore) were cut and glued to a modified 1ml syringe barrel. 300 ⁇ l of 2 x 10 6 /ml neutrophils were added to 300 ⁇ l antagonist and 200 ⁇ l of the suspension was added to the upper chamber (syringe barrel). The lower chamber (a 5ml beaker), contained 3.6 ml of agonist. After 45 minutes, the cells were fixed in 70% ethanol for 5 minutes. This procedure also removes the filter from the syringe barrel by dissolving the glue.
  • the filters were placed in a multi-well dish and treated as follows: distilled water for 2 minutes, Harris haematoxylin 1 minute, distilled water 1 min, Scotts Tap water (1:1 0.7% sodium bicarbonate:4% Magnesium sulphate (v/v) for 5 minutes, 7.0% ethanol 3 mins, 95% ethanol 3 minutes and 80%:20% ethanol:butanol (v/v) 5 minutes.
  • the filters were cleared in xylene for 5 minutes, mounted in DEPEX and examined under bright field illumination with a 40x objective. Five randomly selected fields were counted for each filter.
  • mice were injected subcutaneously in one hind paw with 300 ⁇ g carrageenin mixed the thymosin ⁇ 4, native or sulphoxide in a final volume of 50 ⁇ l.
  • Control animals were injected with the same volume of saline. Footpad swelling was measured using a spring-dial calliper, and expressed as the difference in swelling between the carrageenin-injected paw and the uninjected, contralateral paw.
  • the animals were injected intraperitoneally (i.p.) With the same dose of thymosin ⁇ 4, native or sulphoxide and footpad measurements made at 6, 24, 48 and 72 hours.
  • STMS Steroid treated monocyte supernatant
  • STMS was obtained essentially as described by Chettibi et al. [1993] by the culture of human monocytes in Hams F-10 medium with 10% foetal calf serum (FCS) at a concentration of approximately 5x10 7 cells per ml for 60 minutes, rinsed with PBS, and then cultured without FCS for 24 hours in the presence of 10 -6 M dexamethasone. Parallel cultures in which dexamethasone was omitted were used to prepare control monocyte supernatant (CMS). Purification of STMS peptide factor was carried out using the 2-dimensional diffusion coefficient to identify active fraction. Partial purification was achieved using gel filtration and ion-exchange chromatography on mono-Q resin. Highly purified material was obtained by the additional use of reverse phase HPLC.
  • FCS foetal calf serum
  • STMS Peptide Factor lies in its potential role as a mediator of some or all of the anti-inflammatory effects of glucocorticoids.
  • Many preliminary observations using the supernatant as opposed to the peptide factor seemed to support this role, but others, such as the phenomenon of dispersive locomotion, were not obvious anti-inflammatory responses.
  • lowered adhesiveness which appears to be one of the underlying causes of dispersive locomotion, has clear anti-inflammatory implications.
  • Neutrophils treated with partially purified STMS peptide factor appeared under phase-contrast microscopy to be elongated and the characteristics of their adhesion to a protein-coated glass surface indicated that they might be attached at a single site.
  • the cells were relatively easily detached by washing and also the appearance was suggestive of largely unattached cell body connected to the surface by an elongated process.
  • the cells were of extended bipolar shape but the two ends that appeared to be involved in adhesive contact with the substratum were not identical.
  • the membrane was relatively smooth with numerous small protrusions and there was no very clear difference in surface appearance between the cell body and the apparent pseudopodia.
  • Partially purified peptide factor reduced the adhesiveness of neutrophils to an endothelial cell monolayer. Scanning EM studies of neutrophil/endothelium interactions showed that in marked contrast to other stimuli, the supernatant peptide factor prevents adhesion and apparent invasion at endothelial cell junctions.
  • Partially purified peptide factor inhibited the secretion of elastase from cytochalasin treated neutrophils (data not shown).
  • actin polymerisation is an early response to stimulation of neutrophils by fMLP.
  • Coates et al. [1992] have provided evidence that actin polymerisation is the early and dominant event in determining shape changes and dictating the specific patterns of polarisation.
  • the link between the fMLP receptor and these early membrane events is believed to be provided by protein kinase C (PKC) activation that then leads to activation of the small cytosolic GTP-binding proteins [Ridley, 1994].
  • PKC protein kinase C
  • the pattern of responses to STMS is unusual in many important respects.
  • the actin pool appears to be highly dynamic, but the distribution of F-actin is mainly constrained to one end giving a unipolar distribution of actin in a bipolar cell.
  • the membrane over most of the cell body remains relatively smooth and this lack of ruffling was observed both with cells in suspension and on the surface of cells stuck (albeit loosely) to the endothelium.
  • the lack of ruffles may manifest itself in the inability of these neutrophils to discern the gaps between endothelial cells, thereby inhibiting an inflammatory response.
  • STMS peptide factor as an anti-inflammatory mediator is its modulation of cell responses to pro-inflammatory mediators. This has been tested in the above work using motility stimulation as the basic parameter for comparison of activities. Under conditions where STMS and fMLP or IL-8 are equipotent as locomotion simulators, the morphological, adhesive and locomotor responses to STMS tend to dominate and the cells remain phase-bright, bipolar and undergo dispersive locomotion.
  • STMS Peptide Factor which is not itself chemotactic, is able to suppress the chemotactic response to fMLP. It is of interest here to discuss the role of persistence in chemotaxis. By the very nature of the concept, any form of directed locomotion has persistence. This is seen most clearly in the case of the tilt assay [Chettibi et al., 1994] where the response to STMS showed more than 90% directional movement and the persistence although infinite, became an essentially meaningless concept. For locomotion in a uniform concentration of STMS, persistence reflects some kind of inertia in the locomotor system. Bearing in mind the low Reynolds number conditions, this inertia must relate to the organisation of the structure on which the cytoskeleton acts or redistributes itself during locomotion.
  • the electron micrographs of STMS-treated cells on an endothelial layer show firstly that the cells do not seek the junctions between endothelial cells or do not have suitable exploratory leading lamellae to penetrate such a gap if encountered.
  • STMS Steroid Treated Monocyte Supernatant
  • Dr Chettibi et al. The material described by Dr Chettibi et al. was highly purified by a combination of ion-exchange and gel filtration steps, but the critical procedure was reverse phase HPLC using an HPLC column and an elution gradient made from
  • the most active material prepared by this method is light pink in concentrated solution and was characterised by an elution trace showing an absorption peak at 214nm with a distinct shoulder. Further resolution was not possible and neither could the activity be attributed to the peak or the shoulder. Mass spectroscopy showed a single major component of mass 1331 Da and fragmentation analysis gave species of 1186 Da and 991 Da. Full analysis (Dr Pappin ICRF) confirmed that the major peak was acyanocobalmin, a derivative of vitamin B 12 . It was surmised that vitamin B 12 may in fact be a contaminant or serving to mask the actual peptide factor. Preparation of STMS and subsequent purification of the peptide factor was therefore conducted omitting vitamin B 12 .
  • Mass spectroscopic analysis now revealed a single major peak of average mass 4980 Da (+/- 2 Da). Mass measurement following esterification of a small portion of the peptide material indicated the presence of 11 acidic amino acid residues (Aspartic and Glutamic acid).
  • Peptide was then digested with 100ng trypsin (Boehringer, modified) in 6 ⁇ l 50 mM ammonium bicarbonate (pH 7.8) containing 15% v/v n-propanol and 0.5% hexyl-B-glucopyranoside (HBG) overnight at 25°C.
  • the digested peptides were then reacted with N-succinimidyl-2(3-pyridyl)acetate (SPA) in order to enhance b-ion abundance and facilitate sequence analysis by tandem mass spectrometry (Sherman et al., 1995).
  • Dried peptide fractions were treated with 7 ⁇ l 1% w/v N-succinimidyl-2(3-pyridyl)acetate in 0.5M HEPES (pH 7.8 with NaOH) containing 15% v/v acetonitrile for 20 min on ice.
  • the reaction was terminated by 1 ⁇ l heptafluorobutyric acid (HFBA) and the solution immediately injected onto a capillary reverse-phase column (300 um x 15 cm) packed with POROS R2/H material (Perseptive Biosystems, MA) equilibrated with 2% v/v acetonitrile/0.05% v/v TFA running at 3 ⁇ l/min.
  • HFBA heptafluorobutyric acid
  • the adsorbed peptides were washed isocratically with 10% v/v acetonitrile/0.05% v/v TFA for 30 minutes at 3 ⁇ l/min to elute the excess reagent and HEPES buffer.
  • the derivatised peptides were then eluted with a single step gradient to 75% v/v acetonitrile/0.1% v/v formic acid and collected in a single 4 ⁇ l fraction.
  • CID low-energy collision-induced dissociation
  • Thymosin ⁇ 4 was now prepared from human neutrophils by the method of (Hannapell et al 1982) which used HPLC purification of the perchloric acid supernatant. Four major peaks were obtained and analysed by mass spectroscopy. The identification of two of these peaks was confirmed as Thymosin ⁇ 4 (the major peak) eluting at 37% B and oxidised thymosin ⁇ 4 eluting at 34% B. Two other peaks gave no MW signatures.
  • Thymosin ⁇ 4 was also synthesised by Dr Pappin but the final product had an unidentified modification believed to lie in the C-terminal serine.
  • Dr Pappin showed this molecule to have average mass 4980Da (+/- 2 Da).
  • T ⁇ 4so The activity of T ⁇ 4so, in the neutrophil locomotion assays showed T ⁇ 4so as being dispersive at low concentrations and stimulating non-dispersive locomotion above a concentration optimum, Figure 8a.
  • Native T ⁇ 4 did not give significant dispersive locomotion in these assays.
  • Neutrophil locomotion assays are notoriously hard to standardise and we therefore attempted to confirm the identity of the factor using an independent assay, the inhibition of chemotaxis. Earlier work had shown that STMS inhibited neutrophil chemotaxis to fMLP (Young et al 1997).
  • T ⁇ 4 and T ⁇ 4so were the biologically active extracellular form of T ⁇ 4 and we investigated this by comparing the activity of the two species in the endothelial sheet wound closure test, one of the more accessible bioassays for T ⁇ 4 activity (Malinda et al 1997). The results for the effect of T ⁇ 4 were in close agreement with published data, but T ⁇ 4so was active with at least an order of magnitude higher potency than the native peptide, Figure 8c.

Claims (15)

  1. Pharmazeutische Formulierung umfassend oxidiertes Thymosin β4, eine physiologisch aktive Variante oder ein Salz davon und einen pharmazeutisch verträglichen Träger dafür, worin das aktive Agens das oxidierte Thymosin β4 ist.
  2. Pharmazeutische Formulierung nach Anspruch 1, worin das oxidierte Thymosin β4 eine Form von Thymosin β4 ist, in welcher ein Methionin-Rest, 6 Aminosäuren vom N-Terminus, oxidiert ist zu Methionin-Sulfoxid.
  3. Pharmazeutische Formulierung nach Anspruch 1, worin das oxidierte Thymosin β4 eine Form von Thymosin ist, in welcher ein Methionin-Rest, 6 Aminosäuren vom N-Terminus, komplexiert ist mit Metall(en).
  4. Pharmazeutische Formulierung nach Anspruch 1, worin die physiologisch aktive Variante ein oxidiertes Peptid ist, welches ein Spaltprodukt des oxidierten Thymosin β4 ist.
  5. Pharmazeutische Formulierung nach einem beliebigen vorangehenden Anspruch, worin das oxidierte Thymosin von einern Säuger stammt.
  6. Pharmazeutische Formulierung nach Anspruch 1 bis 4, worin das oxidierte Thymosin β4 synthetisiert ist.
  7. Pharmazeutische Formulierung nach einem beliebigen vorangehenen Anspruch, worin das oxidierte Thymosin β4, eine physiologisch aktive Variante oder ein Salz davon mindestens 30% des Gesamt-Thymosins β4 in der Formulierung ausmacht.
  8. Pharmazeutische Formulierung nach einem beliebigen der Ansprüche 1-6, worin das oxidierte Thymosin β4, eine physiologisch aktive Variante oder ein Salz davon im wesentlichen kein nicht-oxidiertes Thymosin β4 umfasst.
  9. Pharmazeutische Formulierung nach einem beliebigen vorangehenden Anspruch umfassend ein zusätzliches pharmazeutisches Agens, worin eine Nebenwirkung des zusätzlichen pharmazeutischen Agens Entzündung ist.
  10. Pharmazeutische Formulierung nach einem beliebigen vorangehenden Anspruch, die zur oralen, nasalen, topischen, rektalen, vaginalen oder parenteralen Anwendung geeignet ist.
  11. Oxidiertes Thymosin β4, eine physiologisch aktive Variante oder ein Salz davon zur Verwendung als Medikament.
  12. Verwendung von oxidiertem Thymosin β4, einer physiologisch aktiven Variante oder einem Salz davon bei der Herstellung eines Medikamentes zur Behandlung eines entzündlichen Zustandes.
  13. Verwendung nach Anspruch 12, worin der entzündliche Zustand zurückzuführen ist auf eine entzündliche Arthropathie, Kollagenose, Vaskulitis-Syndrom, Atemwegserkrankung, dermatologische Erkrankung, gastrointestinale Erkrankung, hämatologische Erkrankung, Abstossung oder Infektion durch Transplantation oder Prothesen.
  14. Verwendung nach Anspruch 12, worin der entzündliche Zustand das Ergebnis einer separaten Arzneimitteltherapie ist.
  15. Verwendung von oxidiertem Thymosin β4, einer physiologisch aktiven Variante oder einem Salz davon bei der Herstellung eines Medikamentes zur Behandlung von septischem Schock.
EP99913428A 1998-03-28 1999-03-29 Oxydiertes thymosin beta 4 Expired - Lifetime EP1094833B1 (de)

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GBGB9806632.7A GB9806632D0 (en) 1998-03-28 1998-03-28 Peptide factor
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PCT/GB1999/000833 WO1999049883A2 (en) 1998-03-28 1999-03-29 Oxidized thymosin beta 4

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US20070015698A1 (en) * 1998-07-30 2007-01-18 United States Of America As Represented By The Secretary Of Health Treatment of skin, and wound repair, with thymosin beta 4
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WO2002036143A1 (en) 2000-11-02 2002-05-10 Regenerx Biopharmaceuticals, Inc. Inhibition or reversal of skin aging by actin-sequestering peptides
US20070191275A1 (en) * 1998-07-30 2007-08-16 Regenerx Biopharmaceuticals, Inc. Method of treating, preventing, inhibiting or reducing damage to cardiac tissue with thymosin beta 4 fragments
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CN101195025A (zh) * 2001-03-15 2008-06-11 雷金纳克斯生物制药公司 胸腺素β4、类似物、异构体及其它衍生物在制备治疗眼睛和周围组织失调的药物中的应用
JP2005502672A (ja) 2001-08-29 2005-01-27 リジェナークス・バイオファーマスーティカルズ・インコーポレイテッド サイモシンβ４、類似体、アイソフォームおよびその他の誘導体を用いて心筋障害の発生前、発生中または発生後に発生する炎症、損傷およびその他の変化を治癒または予防する方法
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CN101297965B (zh) * 2008-06-16 2011-01-05 浙江省中医药研究院 胸腺肽β4在制备防治支气管哮喘药物中的应用
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